In immunohistochemistry or in situ hybridization reactions, evaporation of the reaction mixture on the slide during incubation periods must be prevented. This is particularly important where a reaction mixture has a small volume, especially when heat is used to control or enhance the reaction kinetics. If evaporation occurs, the concentration of reagents changes, and the tissue section can dry out. Either of those conditions can cause erroneous results.
Traditionally, evaporation is controlled by either placing the slide in a humidified chamber or placing a glass or plastic coverslip over the slide and sealing the sides of the coverslip. Both of those methods are time consuming and cumbersome, and neither can be easily adapted to automation. In addition, use of a coverslip presents a risk that the tissue section will be damaged or removed from the slide when the coverslip is removed.
An improved method described in International Application No. PCT/US91/01108 published Sep. 5, 1991 (Publication No. WO 91/13336) controlled evaporation by covering the aqueous reaction mixture with an evaporation inhibitor liquid which was immiscible in the aqueous phase and had a density less than the aqueous phase. In a preferred embodiment, the evaporation inhibitor liquid was a non-aromatic hydrocarbon having from 6 to 18 carbons, preferably pentadecane (mineral oil). The evaporation inhibitor liquid covered the surface of the reaction mixture, preventing evaporation of the reagents.
This method is applicable to situations in which the ambient temperatures are less than approximately 70.degree. C. However, at temperatures of 70.degree. C. and higher, evaporation of the protective layer of mineral oil results in the eventual loss of protection. This loss can be very rapid (less than 5 minutes) at 100.degree. C. Since certain detection chemistries must be performed at these elevated temperatures (nominal 95.degree. C., range 68.degree. C. to 110.degree. C.), a protective overlay resistant to evaporation at the operating temperatures and having other favorable properties needed to perform the required chemistries is desirable.